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    Home > Biochemistry News > Biotechnology News > Detection method of superoxide disambiguation enzyme (SOD).

    Detection method of superoxide disambiguation enzyme (SOD).

    • Last Update: 2020-10-23
    • Source: Internet
    • Author: User
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    related topics .Abstract:
    soD catalysis destroys superoxygen (O2-) and builds the first line of defense against oxytoxicity. The activity of SOD and its detection technology are closely related to many different fields, such as medicine,
    bio-chemical
    ,
    plant
    , food engineering, etc. In the past few decades, a variety of SOD detection methods have been discovered, but they have their own defects in selectivity, speed, price, and simplicity. Recently, we found that water-soluble tyro-salts such as XTT, WST-1, WST-8 are suitable for O2-testing and can also be used for SOD detection. Among these tyrithic salts, WST-1 has high sensitivity, low absorbent of oxidation and good water solubility, so it is most suitable for SOD detection. This method of testing SOD with WST-1 can be used in bio-chemical samples such as red blood cells, heart and liver in mice. We also found a detection system that uses WST-1 streaming injection to detect SOD, which allows for rapid detection (up to 30 samples per hour).soD detection methods are commonly used in the
    1. phosphorescic method
    phosphorescies method is the most typical method of detecting SOD, which uses cytochrome C (cytochrome C) or nitrogen blue tylutin (NBT) (Figure 1). O2 is detected using the restore method of cytochrome C - because the restored cytochrome C produces purple dyes (Figure 2). This is the most commonly used method since soD was discovered
    Cyt (FeIII.) - > Cyt (FeII.) - O2
    because cytochrome C is easily reduced by NADPH reductase or other reducing agents, so the contamination of the sample must be taken into account. Moreover, this method needs to be looked at every 1.5 minutes and is therefore not suitable for large-volume experiments. The principle of the NBT method is to produce insoluble (max: 560 nm) to go and O2-react. The dye is insoluble in water and produces unearel sediments during long-term analysis, which affects the reproducible nature of the experimental data. In order to obtain water-soluble meth dyes, many improved methods such as
    BSA
    are used. However, adding unnecessary
    protein
    can make data analysis very complex and NBT can be reduced by a variety of reducing agents. This property of NBT is used for the detection of ketamine, which can act as a marker of diabetes and is an intermediate for maillard reactions. The biggest disadvantage of the NBT method is that even if you add too much SOD, you can't get a 100% inhibition rate because NBT reacts directly
    the
    oxidase.1.
    Chemical Luminescence
    method
    Chemical luminescence
    probes for O2-detection
    can also be used for SOD detection, which is divided into two types, one is glossy and the other is a fluorescent derivative (MCLA). This chemical luminescence reaction is highly dependent on pH. For example, glossy fines emit strong chemical light only at pH 9.0 or higher. Therefore, it is not feasible to detect SOD by this method under physiological pH conditions. On the other hand, MCLA can emit strong chemical fluorescence under physiological conditions, so it can be used for the detection of Cu,Zn-SOD activity in the human brain. However, MCLA is not suitable for use as a probe for SOD detection because it not only reacts with O2-reactions, but also with monotonic oxygen. Moreover, MCLA reacts with dissolved oxygen to emit chemical light, and the transferred metal ions accelerate the oxidation reaction..3. Electronic Spin Resonance (ESR)
    spectral
    method
    at room temperature, the O2-ESR signal in the solution can not be detected directly, but can be obtained indirectly by spin capture. The most common trapper is 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Since O2-trapped DMCO produces a special spectra, the ESR method is one of the most specific of the O2-detection methods. However, the second-order velocity constant between dmPO and O2-is relatively low compared to the O2-spontaneous reaction constant, so a large amount of DMPO (e.g. final concentration 0.45M) needs to be added to the solution. In this way, the DMCO used for each trial will cost a lot of money.
    problem with this approach is the need for a more expensive set of ESR equipment in the experiment.4. A new method for detecting SOD using water-soluble tetraazole salts
    In order to overcome many of the above problems, we need to consider the following aspects when doing SOD testing: experimental equipment should be simple, economical, pH sensitivity is low, highly specific to O2-. If
    10th 10th 1st 1st 1st 2000
    is used as a simple experimental device, a color-matching probe similar to cytochrome C and NBT is required. The most important point in the test is to determine the suppression rate of SOD 100% without interference from other substances. In order to obtain a new method of detecting SOD, we have carried out a series of experiments onsalts that can produce water-soluble methamphetalide.4. 1 XTT
    XTT is a water-soluble tetrate first reported in 1988 and has since been used as a substrate of an electronic transfer system for bacteria or mammalian cells. Its structure is shown in Figure 1. NBT has a double tee salt structure, while XTT has a single tee salt and two acid groups.Figure 1. Structure of tyroline in SOD detectionWe use XTT to detect SOD, as shown in Figure 2, with the increase in SOD concentration, a 100% inhibition rate can be observed. This 100% suppression rate has never been obtained in the past with the NBT method. A 100% suppression rate can be observed even under different pH conditions. This 100% suppression rate of XTT means that XTT will only be restored by O2-specifically, and XTT solves problems that NBT cannot solve.2. The suppression curve of SOD obtained by NBT (-) and XTT (-0)
    .
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